J AC Zinc ion availability the determinant of efficacy in zinc lozenge treatment of common colds

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1 85. Peter, G., Chernow, M., Keating, M. H., Ryff, J. C. & Zinner, S. H. (1982). Limited protective effect of rough mutant antisera in murine Escherichia coli bacteremia. Infection 10, Trautmann, M. & Hahn, H. (1985). Antiserum against Escherichia coli J5: a reevaluation of its in vitro and in vivo activity against heterologous Gram-negative bacteria. Infection 13, Appelmelk, B. J., Verweij-van Vught, A. M. J. J., Maaskant, J. J., Schouten, W. F., de Jonge, A. J. R., Thijs, L. G. et al. (1988). Production and characterisation of mouse monoclonal antibodies reacting with lipopolysaccharide core region of Gram-negative bacilli. Journal of Medical Microbiology 26, Baumgartner, J.-D., Heumann, D., Gerain, J., Weinbreck, P., Grau, G. E. & Glauser, M. P. (1990). Association between protective efficacy of anti-lipopolysaccharide (LPS) antibodies and suppression of LPS-induced tumor necrosis factor and interleukin 6. Journal of Experimental Medicine 171, Received 7 January 1997; accepted 30 April 1997 J AC Zinc ion availability the determinant of efficacy in zinc lozenge treatment of common colds Journal of A ntim icrobial Chem otherapy (1997) 40, George A. Eby* G eorge E by R esearch, 2109 Param ount A venue, A ustin, T X 78704, USA This is a re-analysis of reports from 1984 to 1992 of double-blind, placebo-controlled, clinical trials of zinc lozenges in the treatment of common colds. This re-analysis was performed to test the hypothesis that major variations in daily zinc ion availability (ZIA) between chemically different lozenge formulations caused differing results in these clinical trials. Solution chemistry computations determined the bioavailability of Zn 2+ ions at physiological ph from the lozenges used in these clinical trails. ZIA was derived from Fick s laws of diffusion in a bio-electric field. Lozenges that released Zn 2+ ions at physiological ph (positive ZIAs) shortened colds. Lozenges that released negatively charged zinc species at physiological ph (negative ZIAs) lengthened colds. Lozenges having a zero ZIA had no effect on common colds. Lozenges with ZIA = 100 shortened colds by 7 days while ZIA = 55 lozenges lengthened colds by 4.4 days. A linear dose response relationship exists between ZIAs of zinc lozenges and changes in duration of common colds. It is concluded that: prospective efficacy of zinc lozenges can be predicted based upon readily determined ZIA factors and ZIAs; chemically different zinc lozenge formulations having greatly different ZIAs resulted in greatly differing results in clinical trials; mast cell granule-derived Zn 2 + ions are the foundation of the primary immune system; and high ZIA zinc acetate lozenges are beneficial for common colds. Introduction In 1984, the present author and co-workers 1 reported that lozenges releasing 23 mg zinc (from zinc gluconate) shortened the duration of common colds by an average of 7 days in a double-blind, placebo-controlled clinical trial. No other soluble excipients were present in the unsweetened lozenges. The original report was confirmed by A l-nakib et al. 2 at the Medical Research Council (MRC) Common Cold Unit in 1987 using 23 mg of zinc from zinc gluconate in flavoured fructose tablet. O ther clinical research using zinc gluconate and food acid additives, low zinc gluconate dosages and other zinc compounds was published from 1987 to These r e p o r t s showed conflicting results. Potter & Hart 3 r e v i e w e d all the published literature in U nfortunately, they did not consider (i) solution chemistry analyses of the lozenges used in the reviewed trials, (ii) unpublished data available only from lozenge manufacturers and authors of the original reports and (iii) analyses of the bioavailability of Zn 2 ions to the oral mucosa at physiological ph. This re-analysis includes that omitted information, and shows that the bio- *Tel: 1-(512) ; Fax: 1-(512) ; coldcure bga.com 1997 The British Society for A ntimicrobial Chemotherapy 483

2 G. A. Eby availability of Zn 2 ions at physiological ph is associated with positive results and that the absence of bioavailable Zn 2 ions at physiological ph is associated with negative results. Common cold aetiology New data obtained by polymerase chain reaction techniques indicates that about 60 70% of upper respiratory viral infections (common colds) are caused by rhinoviruses. 4 Common cold symptoms are generally believed to be caused by over-reaction of the immune system to these relatively harmless viruses. In-vitro effects of Z n 2 ions on rhinoviruses Zn 2 ions at a 0.10 mm concentration were demonstrated by Korant, Butterworth and their co-workers 5 9 to be highly effective antirhinoviral agents inhibiting cleavage of rhinovirus polypeptides in vitro. Merluzzi et al. 10 showed that the effects of zinc on rhinovirus replication were related to the concentration of Zn 2 ions and unrelated to the total amount of zinc. They also showed that the antirhinoviral effect of Zn 2 ions was as strong as the antirhinoviral effect of interferon at its most effective concentration 10 No invitro effects were found by Geist et al. 11 below 0.03 mm, a finding in agreement with those of the others Other researchers have found that Zn 2 ions at about 0.10 mm induce the production of large quantities of interferon and are mitogenic to T-cell lymphocytes in vitro Changes in cell appearance suggested toxicity of Zn 2 ions to some authors, 10,11 but many others strongly assert that Zn 2 ions lack cytotoxicity and that, rather, they cause potent stabilization of cell membranes, drying effects, astringency and anti-inflammatory action A stringency versus pseudo-astringency Zn 2 ions are well known for their astringency. Pasternak 15 has shown that elevated Zn 2 ion serum concentrations are a form of host defence acting to stabilize and protect cell membranes. All astringents, including Zn 2 ions, are locally applied protein precipitants having such low cell penetrability that their action (contraction, rounding and blanching in vitro) is essentially limited to cell surfaces and interstitial spaces. Permeability of cell plasma membranes is reduced by astringents, including Zn 2, but the cells remain viable. A stringents harden the cement substance of capillary epithelium, inhibiting pathological transcapillary movement of plasma proteins, and reduce local inflamma - tion, oedema, and exudation. A dditionally, astringents reduce mucus and other secretions in tissues containing goblet cells and other secretory cells, causing affected areas to dry and heal faster. 16 Pseudo-astringents include anionic metal complexes in solution, acids, alcohols, phenols and other substances that precipitate proteins. They may seem astringent to the mouth but they readily penetrate cells and promote tissue damage. 16 Food acid flavoured zinc lozenges caused sufficient oral tissue irritation in several trials to imitate the oral tissue irritation reported in the successful studies by Eby et al. 1 and Al-Nakib et al. 2 and this pseudo-astringency incorrectly convinced several common cold researchers of the availability of zinc ions from their research lozenges. O nly analysis by solution chemistry methods enables the correct determination of availability of metallic ions at physiological ph. Clinical trials re-analysed Contact with lozenge manufacturers and clinical researchers to obtain critical missing lozenge solution chemistry data and trial data was essential in order to provide a valid re-analysis. A ll clinical trials re-analysed h e r e 1, 2, used accepted, double-blind, placebo-controlled, clinical research methods. A ll known reports of zinc lozenges as therapy for common colds are included. With the exception of two clinical trials, 26,28 all patients were prohibited from using other common cold medications. No patients suffering from allergies or other diseases or conditions that might mislead the results were included in these studies. No trial permitted enrolment of pregnant women. All but two trials 2,29 reported results from natural colds. The demographics of patients in each trial were found to be well randomized. The number of patients in the clinical trials varied from 55 to 146. Methods The zinc ion availability (Z IA) method of analysis was developed to evaluate the amount of Zn 2 ions available at physiological ph for absorption into the oral and oropharyngeal tissues, and to determine if the in-vivo dose response to Zn 2 ions was similar to the in-vitro dose responses shown by Merluzzi et al. 10 The review also evaluated the hypothesis that Zn 2 ions in saliva diffuse into the infected tissues of the nose from the membranes of the oral cavity via oral nasal tissues. Fick s laws of diffusion were used to derive both the ZIA method of a n a l y s i s and diffusion theories. Fick s laws of diffusion Fick s first law of diffusion is dm/dt DA dc/dx, where m is the quantity of drug or solute diffusing in time t, dm/dt is the rate of diffusion, D is the diffusion constant, A is the cross-sectional area of the membrane, dc is the change in concentration, and dx is the thickness of the membrane. 30 Fick s second law of diffusion is similar and relates to diffusion through solids. The rate of drug transport over time can be altered by a change in any of the above variables. If both the cross-sectional area of the oral cavity and the rate 484

3 of mouth-to-nose diffusion is held constant and lozenge dissolution times are introduced, ZIA can be calculated. 26 Z inc ion availability Z inc ion availability is the term used to identify the potential for absorption of Zn 2 ions at physiological ph 7.4 into oral and oropharyngeal mucosal membranes. It is given by ZIA KZ i T, where K is (the constant used to set ZIA value from the Eby et al. trial 1 to the reference value of 100), Z i is the initial concentration (mm) of Zn 2 ions and T is the daily duration of oral contact time. For comparative purposes, Z IA can be determined by multiplying the constant by the zinc dosage (mg) in the lozenge the fraction of zinc available as Zn 2 ion at ph 7.4 the oral dissolution time (min) of each lozenge the number of lozenges used per day, and dividing by the volume of saliva (ml) generated during each oral dissolution. For example, the ZIA calculation for the lozenges used by Eby et al. 1 is: (mg zinc/lozenge) 0.30 (fraction of zinc as Zn 2 ion at ph 7.4) 30 (dissolution time in minutes) 9 (doses per day), divided by (ml saliva which numerically equals the total saliva weight minus lozenge weight). Specific gravity is considered for soluble lozenges. Initial Zn 2 ion concentrations are times greater than the concentration needed to have pharmacological activity in vitro. For example, the successful Eby et al. 1 lozenges produced a salivary Z i concentration of 7.4 mm, while the pharmacologically active in-vitro concentration is only 0.1 mm. However, many Zn 2 ions are lost during their diffusion through tissues from interactions with diverse ligands found in saliva, blood, lymph and tissues. stability constant for the metal and its associated ligand. The basic solution chemistry equation, [CM] [M] [ML], where [CM] is the total metal concentration, [M] is the concentration of metallic ions and [ML] K [ML][L] (where [L] is the free concentration of ligand and K is the stability constant of the metal ligand complex), results in the equation [CM] [M](1 K[L]). Consequently, [M] [CM]/(1 K[L]), which shows that [M] depends upon [CM], K and [L] which in turn depend upon corresponding pk and ph values (personal communication, G. Berthon, D irector of R esearch, INSE R M, U-305, Toulouse, France). Figure 1 shows that 30% of the zinc from zinc gluconate is available as Zn 2 ions at physiological ph. 33 Because of the extremely low affinity of sweet carbohydrates for Zn 2 ions, 34 Figure 1 also applies to aqueous solutions of most sweet carbohydrate-based zinc gluconate lozenges. Figure 2 shows an absence of Zn 2 ions at each ph greater than 6 in the system with a 1:1.33 ratio of zinc gluconate to citric acid and the presence of negatively charged zinc citrate species at physiological ph. Figure 3 shows an absence of Zn 2 ions at each ph greater than 6 in the system with a 1:10 ratio of zinc gluconate to glycine and presence of mainly neutrally charged zinc glycinate at physiological ph 7.4. Similarly, the bioavailability of Zn 2 ions for other commonly used zinc compounds is shown in Figure 4. M easuring com m on cold treatm ent responses H alf-lives of common colds and weighted average durations of exponentially decaying common colds have been Physiological ph The only relevant ph the for purposes of determining the availability of Zn 2 ions in tissue, lymph and blood is physiological ph 7.4. This results from all acids and bases being instantly buffered by the bicarbonate, phosphate and protein buffer systems in tissue, lymph and blood during homeostatic regulation of acid base balance. 31,32 The Zn 2 ion is a Lewis acid capable of existing at physiological ph Salivary ph was 5.5 when zinc gluconate lozenges having no other zinc ligands were used. Salivary ph ranged from ph 4.3 to 7.0 in lozenges containing other zinc salts. 26 In instances when the salivary or infected respiratory secretion ph is lower than physiological ph, Zn 2 ion concentration is higher. H owever, these higher concentrations are reduced as a result of being instantly buffered to ph 7.4 upon absorption. 26 B ioavailability of m etallic ions Bioavailability of metallic ions, including Zn 2 ions, occurs only at physiological ph 7.4 and is dependent upon the first Figure 1. Distribution of zinc ionic species in the Zn 2 and gluconic acid (L) system. Curves were constructed from pk values shown after the reactions: Zn 2 L ZnL (1.62) and ZnL OH ZnL(OH) 0 (8.14) at a concentration of 30 mm zinc. The pk values are courtesy of Gerritt Bekendam, A kzo Chemicals BV Research Centre, Deventer, The Netherlands. The Zn 2 fraction over ph 6 is strongly affected by the second pk value. Precipitates of hydroxides of zinc result in supersaturated solutions above ph 7.4. (Figure reproduced with permission. 33 ) 485

4 G. A. Eby Figure 2. Distribution of Zn 2 species plotted against ph for solution with Zn 2 and excess citric acid (H 3 L). At any ph, the curves sum to unity. Curves were constructed from the stability constant logarithms in parentheses after each reaction: Zn 2 HL 2 ZnLH (3.0), Zn 2 L 3 ZnL (4.8), and ZnL L 3 4 ZnL 2 (1.7). Except for the ratio of the last two complexes, the general shapes of the curves are independent of the specific concentrations of 18 mm Zn 2 and 23 mm citric acid. Successive pk a values for citric acid deprotonations are pk 1 3.0, pk and pk Not included in the analysis is a small amount of ZnLH 2 of low stability likely to form near ph 3. Figure reproduced with permission. 38 Figure 3. Distribution of Zn 2, positively charged zinc gluconate (ZnL ), and various zinc glycinate and zinc hydroxide species in the 1:10 M zinc gluconate glycinate system. Zinc and gluconate are present at 30 mm and glycine is present at 300 mm concentration. Distribution of zinc ionic species is courtesy of G. Berthon, INSERM U 305, CNRS, Toulouse, France. 26 shown to be acceptable analytical mechanisms. 1 Half of untreated rhinovirus colds resolve in 1 week, threequarters resolve in 2 weeks, while seven-eighths last 3 weeks or less, 35 which indicates an exponential decay rate, with a half-life (T 1 ) of 7 days. By integrating, the average duration is T 1 /ln 2. 1,26 If the observed T 1 of untreated colds is 7 days, then the calculated average duration of those same colds is 10 days. The accepted model for calculating treatment efficacy is to subtract the T 1 of actively treated Figure 4. Percentage of zinc released as biologically available Zn 2 ions at ph 7.4 from zinc compounds used in common cold clinical trials plotted against the logarithm of the first stability constant K 1. colds from the T 1 of placebo-treated colds, and likewise for the average duration of colds. Methods of obtaining the average duration of colds differed slightly among trials because the researchers methods of reporting were not uniform. Average duration data and half-life data were published for the Eby et al. zinc gluconate trial. 1 Mean clinical score end-points were used to calculate the average duration of colds in the Al-Nakib et al. zinc gluconate trial. 2 In the Smith et al. zinc gluconate trial, 24 average durations were calculated from half-lives obtained from published figures and data. A verage durations of colds were stated by the authors in the Weismann et al. zinc gluconate, 2 5 Eby et al. zinc orotate, 2 6 M c C u t c h e o n et al. zinc aspartate, 26 Douglas et al. zinc acetate tartarate carbonate, 27 and Godfrey zinc gluconate glycine 28 trials. In the case of the zing gluconate glycine trial, 28 this reanalysis showed that the claimed reduction in duration was erroneously reported, because data were obtained by sub - tracting observed half-lives of colds from historic average duration of colds. Average duration of colds in the zinc gluconate citrate trial 29 were determined from day 7 symptom scores. L oz enge side-effects and safety D ocumented side effects and safety data were taken directly from the reports of clinical trials of zinc lozenges. Statistical methods Statistical methods used in each trial s research report were cited as originally reported. Spearman s rank difference correlation method was used in analysis of the relationship between lozenge Z IA values and their effects on common colds. 486

5 Figure 5. Relationship of zinc ion availability (ZIA) values and reduction in duration of common colds in days The regression equation for non-negative values is y 0.077x Straight line projection using this equation and increases in duration data provided estimates for negative ZIAs. There is no 1:10 M zinc gluconate glycine data point 28 because it is a horizontal line in the upper left quadrant. Results The main finding of this re-analysis is the dose response linearity shown in Figure 5. E vidence of dose response linearity is sufficient to reject the null hypothesis that there is no relationship between ZIAs and changes in duration of common colds. ZIAs not total zinc provide a means of evaluating zinc lozenges of different formulations. Each of the reports listed in Tables I and II and represented in Figure 5 is briefly summarized below. In the original double-blind, placebo-controlled clinical trial by Eby et al., 1 23 mg zinc (175 mg zinc gluconate) unsweetened lozenges reduced the duration of natural colds by an average of 7 days compared with placebo. The reduction in average duration of colds was highly significant (P 0.10 at 12 h, P at 24 h, and P at day 7). These zinc gluconate lozenges were assigned a ZIA of 100 and hence, using the equation ZIA KZ i T, the value of the constant K, was determined to be A l-nakib et al. reported the effects of pleasant-tasting, highly flavoured, 23 mg zinc (175 mg zinc gluconate), fructose-based lozenges. A s a treatment, zinc gluconate lozenges shortened experimentally-induced rhinovirus-2 colds by a statistically significant average of 4.8 days compared with placebo. Although Zn 2 ion release was the same as in the 1984 Eby et al. trial, 1 the Al-Nakib et al. lozenges dissolved faster and, thus, these zinc gluconate lozenges had a calculated ZIA of 44. In sharp contrast, Smith et al. in reported the effect of bitter, 11.5 mg zinc (87.5 mg zinc gluconate) sucrose-, fructose-, mannitol- and sorbitol-based lozenges in a double-blind, placebo-controlled, clinical trial of natural colds in 174 patients. On day 7 there were 12% fewer ill patients in the zinc-treated group than in the placebo group (P 0.09), and the severity of zinc-treated colds on days 5 7 was reduced by 8% (P 0.02). Compared with placebo, the average duration of common colds was reduced by 1.6 days. These mildly astringent lozenges had a calculated ZIA of 25 because of low zinc gluconate content, rapid lozenge dissolution and bitterness-induced deviation from the clinical trial protocol. In 1990, Weismann et al. 25 found no reduction in common cold duration from very low dose zinc gluconate lozenges in a double-blind placebo-controlled trial of natural colds involving 130 patients in D enmark. These non-astringent, maltitol-based, hard-boiled candy lozenges had a low ZIA of 13.4 because they contained only 4.5 mg zinc (31.3 mg zinc gluconate). Very low zinc dosage was used in an effort to avoid the extreme bitterness of zinc gluconate in maltitol-based lozenges. Non-astringent 37 mg zinc (zinc orotate) lozenges in conjunction with 10 mm zinc gluconate nasal spray were found to have no effect on the duration of colds in a 1984 doubleblind, placebo-controlled trial of natural colds in 77 patients by Eby et al. 26 At physiological ph 7.4, zinc orotate is an insoluble, non-ionizable compound having a first stability constant of log K at 25 C. 36 The ZIA was zero because no Zn 2 ions were released. Non-astringent 24 mg zinc (zinc aspartate) lozenges and paracetamol produced no significant differences in responses compared with placebo and paracetamol in a double-blind trial of natural colds in 100 patients (personal communication, M. L. McCutcheon, Student H ealth D irector, University of Minnesota, Duluth, MN, USA). 26 Zinc aspartate tastes pleasant and has a log K 1 at 37 C of near Because of the protonated ammonium group, the 487

6 G. A. Eby Table I. Zinc ion availability (ZIA) factors 26 Fraction Z inc of zinc D issolution Total Lozenge Trial lozenge/reference dosage a as Zn 2 time b Doses saliva d weight e (formulation) (mg) at ph 7.4 (min) per day c (ml) (g) Zinc gluconate 23 mg (non-soluble compress) Zinc gluconate 23 mg Zinc gluconate 11.5 mg (soluble multi-ingredient compress) Zinc gluconate 4.5 mg (soluble maltitol candy) Zinc orotate (non-soluble compress) Zinc aspartate (soluble multi-ingredient compress) Zinc gluconate-citrate (soluble corn syrup sucrose candy) Zinc acetate tartarate carbonate (soluble mannitol compress) Zinc gluconate glycine (soluble corn syrup sucrose candy) a Amount of zinc per lozenge. b Mean time required for lozenge to dissolve in the mouth. Oral dissolution times of lozenges are considerably greater than those found using USP disintegration tests. c Mean number of doses actually taken, rather than intended number. d Total volume of saliva generated from use of a lozenge. e Actual weight of the lozenges tested. Specific gravity of soluble lozenges is 1.5. Table II. Trial lozenges, ZIA, Zn 2 ion concentration, electronic charge of zinc, and efficacy of treatment 26 m/mol Zn 2 Electronic ions at charge of Change in ph 7.4 zinc species duration of Trial lozenges/reference ZIA (Z 1 ) at ph 7.4 common colds Zinc gluconate 23 mg ,1,0 7 day reduction Zinc gluconate 23 mg ,1,0 4.8 day reduction Zinc gluconate 11.5 mg ,1,0 1.6 day reduction Zinc gluconate 4.5 mg ,1,0 none Zinc orotate none Zinc aspartate none Zinc gluconate citrate a 0 0, N 1 day increase Zinc acetate tartarate carbonate a 0 0, N 4.4 day increase Zinc gluconate glycine 28 Indeterminate 0 0, N 1.27 day reduction a Negative ZIA values were estimated using increase in duration data and straight-line projection of non-negative values using formula shown in Figure 5. conditional first stability constant is log K at ph The high stability constant prevented significant Zn 2 ion release at ph 7.4; therefore, Z IA was barely positive. Zinc gluconate (23 mg zinc) candy lozenges with 90 mg citric acid (1.33 moles citric acid to 1 mole zinc gluconate) as a flavour-mask were tested by Farr et al. in 1987 in trials of clinically induced human rhinovirus-13 and -39 colds in 55 individuals. 29 Compared with placebo lozenges, zinc 488

7 gluconate citrate lozenges worsened cold severity and lengthened them by a day. The log K 1 of zinc citrate is 4.7 at 37 C. 38,39 Zinc, lightly bound to gluconate, readily dissociates and strongly and instantly binds to citrate. Figure 2 shows that the reaction produces only negatively charged zinc species at physiological ph ,39 Pseudo-astringency and a mild acidic taste were caused by anionic zinc species at physiological ph 16 and the ZIA was estimated to be 11. Douglas et al. 27 reported that effervescent zinc acetate lozenges containing 10 mg zinc increased the average duration of natural colds by 4.4 days in 70 patients. Several hundred milligrams of tartaric acid and sodium bicarbonate were included to produce effervescence (personal communication, R. J. E. Williams, Development Scientist, F. H. Faulding & Co., A delaide, A ustralia). Tartaric acid was present in considerable excess relative to zinc, and it has a high log K 1 of Zinc dissociates from acetate and binds instantly to tartarate. Few Zn 2 ions remain available at physiological ph 7.4 and negatively charged zinc species predominate. A nionic zinc species caused pseudo-astringency. The Z IA for tartarate- and carbonate-complexed zinc acetate lozenges was estimated to be 55 because of negatively charged zinc tartarate species. Godfrey et al. 28 reported results of a double-blind, placebo-controlled, clinical trial involving 87 patients. Active lozenges contained 23 mg zinc with 10 mol glycine/ mol of zinc gluconate as flavour-mask in 4.5 g sucrose and corn syrup hard-boiled candy lozenges. Both active- and placebo-treated groups were also given paracetamol. R e- analysis of data and figures provided by the authors suggests a reduction in the mean duration of natural common colds by 1.27 days by zinc compared with placebo. 28 Published results calculated the reduction in mean duration of colds incorrectly by comparing half-lives of colds with the historical mean duration of colds. Due to this error, the authors conclusions that the lozenges shortened the mean duration of colds by 42% and the placebo was efficacious, are incorrect. Log K 1 values for zinc glycine complexes are slightly higher than for zinc citric acid: 4.8 at 37 C, 41 and 4.7 at 37 C. 38,39 Figure 3 shows that zinc is largely dissociated from gluconate and bound to glycine in this 1 M zinc gluconate and 10 M glycine solution. Only neutral zinc glycinate, minor amounts of negatively charged zinc glycinate species and traces of negatively charged zinc hydroxide exist at ph 7.4. Because of these computational errors and the lack of positively charged zinc species at physiological ph, a ZIA cannot be determined. Z IA s ZIAs derived from the clinical trials are shown in Table II. The initial concentration of Zn 2 ions at ph 7.4 (Z i ), electronic charges of zinc species at ph 7.4, and treatment efficacy of lozenges are presented in Table II. Details of Z IA calculations, previously unpublished manufacturers lozenge formulas, methods and manufacturing procedures of lozenges having a major impact upon lozenge ZIAs, and other data too complex or lengthy for presentation in this re-analysis have been published elsewhere. 26 D ose response linearity between Z IA s and com m on cold duration Non-negative data from Table II were analysed to ascertain the correlation between Z IA and reduction in common cold duration. The reduction in duration (response) and Z IA (dose) were linearly related with 0.96 (P 0.02, two-tailed test). R egression analysis of these data points yielded the equation y 0.077x 0.16 as shown in Figure 5. Safety of zinc lozenges None of the zinc or placebo lozenges were harmful. Side effects varied with each clinical trial due to differing formulations. 1,2,24 29 Discussion The central finding of this report is linearity in the dose response relationship between studies using zinc lozenges having positive Z IA s, when Z IA is used as a measure of zinc dosage and the duration of symptoms of common colds as the response. Linearity demonstrates that, through consideration of Z IA, divergent results are completely reconciled and the correlation is consistent with the findings of Merluzi et al. 10 of activity of Zn 2 ions against rhinovirus replication in vitro. A lthough the linear relationship between Z IA and clinical efficacy of trials is clear, the evidence for efficacy relies upon three zinc gluconate trials. 1,2,24 Null results from trials of lozenges having low, zero, and negative Z IA values show that Zn 2 ions are needed for efficacy, and these findings are not in conflict with positive findings. Indeed, they clearly support the in-vivo Zn 2 ion antirhinoviral activity theory presented by Eby et al. 1 and they strongly support the theory that lozenges containing any zinc compound having the same ZIA value will have the same effect on the duration of common colds. The zinc gluconate lozenges having a molar proportion of glycine tested by the Cleveland Clinic Foundation were reported in 1996 to reduce the T 1 of natural common colds by about one-half. 42 Unpublished zinc speciation calculations by Guy Berthon showed that most zinc was in the form of zinc glycinate 1 at physiological ph. Using Figure 5 and the relationship between the T 1 of common colds and their average duration, a ZIA of 70 for these lozenges can be deduced when used six times per day. Prasad, in his accompanying editorial, 43 suggested that the ligand 489

8 G. A. Eby gluconate was the likely cause of bad taste associated with zinc gluconate lozenges, and such problems might have been avoided had zinc acetate been used instead. G enerally, carbohydrates do not bind zinc. For example, dextrose has a first stability constant for zinc of log K This lack of stability results in essentially no competition by dextrose as a zinc binding agent compared with gluconate (log K ) or acetate (log K 1 1.0). 44 However, zinc chloride and dextrose have identical first stability constants, resulting in reactions that discolour white lozenges. On the other hand, zinc chelators, also known as metal binding agents or sequestrants, tightly bind Zn 2 ions at physiological ph. Zinc ions are consequently rendered biologically unavailable if zinc chelators are present in sufficient amount. D ependent upon the amount, ascorbic acid, citric acid, EDTA, salicylic acid, tartaric acid, amino acids and other food acids, acacia (gum arabic) and other vegetable gums, alkalis and their carbonates, oxalates, phosphates, sulphides, caustic lime, lake food colours, histamine, histidine, proteins, porphyrins, peptides and other macromolecules can be incompatible with release of Zn 2 ions from zinc compounds at physiological ph. 45,46 A ll water-soluble anionic chemicals bind zinc to some extent and should be excluded from zinc lozenges. The validity of negative estimates of ZIA might be questioned, but the highly significant fact remains that lozenges releasing negatively charged zinc species (ZnL N ) increased the duration of common colds in a dose response manner compared with placebo. Negative ZIAs are possible only because Zn 2 ions are released by the human immune system mast cell granules in their natural fight against viruses that cause common colds. In mast cells, which are plentiful in the respiratory tract, and basophil granules, Zn 2 ions are highly concentrated (4 20 mm) and unsequestered. 47 In inflammation Zn 2 ions are released during degranulation of these cells. 48 ZnL N from lozenges preferentially bind with positively charged substances, primarily Zn 2 ions released from these cells, and neutralize them to a zero electrical charge. If release of Zn 2 ions from cells during inflammation in common colds inhibits viral replication, 5 10 stabilized cellular plasma membranes, activates T-cells, catabolizes histamine, 37,40 regulates mast cell homeostasis 48 and stimulates massive interferon production as they do in vitro, then one must infer: (i) common colds would be worsened by ZnL N by neutralization of native mast cell originated Zn 2 ions to a neutrally charged, biologically useless species at physiological ph, and (ii) mast cell derived Zn 2 ions are important in the mucosal primary immune system. Therefore, Zn 2 ions from ZIA-positive lozenges would amplify natural immunity and limit the duration of common colds. A s rhinovirus infection occurs in the nose, logic might suggest that Zn 2 ions should be applied to the nose using nose drops or nasal sprays, not to the oral mucosa by lozenges or to the stomach by pills. Furnace analysis atomic absorption spectrophotometry demonstrated that zinc was not found in nasal mucus as result of zinc gluconate lozenge oral dissolution (personal communication, J. M. G waltney, U niversity of Virginia School of Medicine, VA, U SA) suggesting lozenge inactivity and, perhaps, that nose drops would be preferable to lozenges. Since 1903, a range of zinc compounds have been used intranasally as a mild, short-acting decongestant but have shown no evidence of a reduction in the duration of common colds. Z inc gluconate nasal spray used every min in conjunction with zinc orotate lozenges has been reported to have a decongestant effect but no effect upon the duration of the common cold. 26 Frequent nasal application of Zn 2 ions (0.1% w/v zinc sulphate) did not reduce the duration of the common cold but did show a mild decongestant effect (personal communication, D. Bryce- Smith, U niversity of R eading, U K). Thus, more than exposing nasal tissue to Zn 2 ions must be involved in reducing the duration of common colds. The transport of metallic ions long distances through naturally occurring biologically closed electrical circuits (BCE Cs) was described in great detail by B. E. W. Nordenström of the Karolinska Institute in Stockholm in A mong his findings, Nordenström showed that electrical potentials unrelated to nervous system electrical activity are generated by infected, injured, and cancerous tissues as well as healthy muscles. Nordenström also showed that metallic ions adhered to the inside of capillaries, thus changing the charge of capillary walls from negative to positive, thereby providing a conduit for other positively charged metallic ions to move long distances. H e also demonstrated that Fick s laws of diffusion apply for metallic ions in a bio-electric field. Nordenström s observations suggested the existence of a BCEC between the mouth and nose as a possible explanation for the Zn 2 ion lozenge effect on colds. Evidence of a BCEC was found when an mv potential difference was observed between the interior of the mouth and the interior of the nose in 19 unrelated, healthy volunteers.26 Placement of one electrode of an ohmmeter on to an oral cavity surface and the other on to a nasal turbinate usually registered either a 10,000 reading or a reading over 15,000, dependent only upon electrode placement (polarity). The lowest resistance values found were 1000 and 1500 in an individual with chronic nasal drainage and frequent colds, while an individual extremely resistant to upper respiratory infections had the highest mouth nose differential resistance readings of 50,000 and 100,000. A significant resistance change always resulted when electrodes were reversed, mimicking a diode effect. E lectron flow produced by the ohmmeter was either retarded or assisted by the electron flow in the mouth nose BCEC producing the different resistance readings. The mouth nose BCEC moves Zn 2 ions through the oral mucosa and into the infected superficial columnar cells of the nasal turbinate epithelium and nasopharynx, result - 490

9 ing in efficacy from oral cavity Zn 2 ion application. Con - versely, inefficacy of nasal administration of Zn 2 ions is due to the outflow of electrons from the surface of nasal tissue as well as outflow of nasal mucus and action of cilia. Is Prasad 43 correct in suggesting that zinc acetate should be preferred over zinc gluconate? The log K 1 of zinc acetate is Hacht & Berthon 50 demonstrated that 100% of zinc acetate is released as Zn 2 ions at any ph between 2.8 and well above 7.4. In addition, zinc acetate is sufficiently stable to be non-reactive with sweet carbohydrates. Korants demonstrated the rhinovirus replication inhibition properties of zinc acetate in A necdotal evidence evinced by strong repeat purchases of Z IA 100 zinc acetate lozenges in the U SA supports Prasad s suggestion. H owever, only carefully conducted clinical trials will prove or disprove efficacy of zinc acetate lozenges against common colds, and those trials are of immediate international importance. U nlike some unhelpful zinc gluconate lozenges, zinc acetate lozenges are flavour-stable and do not become bitter regardless of time or storage conditions Properly prepared Z IA 100 zinc acetate lozenges have no objectionable taste or aftertaste in common cold treatment. They do not seem to produce side-effects previously associated with zinc gluconate lozenges, perhaps because much less zinc acetate is required to produce identical results than zinc gluconate or any other suitable zinc compound. 26 O ne simple way to determine a priori the likely efficacy of commercial zinc lozenges is to taste-test them. A stringent lozenges suggest efficacy is likely. H owever, oral astringency is less noticeable in people with colds than in well people. This difference could be due to oral, facial and nasal tissues being more permeable in illness, resulting in more rapid absorption and removal of Zn 2 ions from the oral mucosa. On the other hand, an acidic taste usually from citric or ascorbic acid in some commercial zinc gluconate lozenges available throughout the U SA in 1997 is now being associated anecdotally with worsened colds, suggesting a negative ZIA. Additionally, there are zinc gluconate lozenges on the market in the USA that have very low positive ZIAs. Other commercial lozenges containing zinc aspartate have a very low positive Z IA and have essentially negligible effect on common colds. Variations in clinical trial results reviewed above have previously been attributed to placebo unblinding and faulty positive results. The necessity of preventing placebounbinding in clinical trials cannot be overstated. Complete details of taste testing results and placebo matching statistics must be published along with clinical results for positive results to be convincing. Clinical trials must be conducted in a statistically valid manner as demonstrated by Gwaltney et al. 54 A cknowledgements Special thanks are extended to Dr Guy Berthon, Director of R esearch CNR S, INSE R M U nit-305 E quipe Bioréactifs: Spéciation et Biodisponibilité, Toulouse, France, for definitive determinations of zinc speciation for several important zinc compositions. Special thanks are also extended to Dr Ananda S. Prasad, Director of Research, D epartment of Internal Medicine, Wayne State U niversity School of Medicine, Detroit, MI, USA; Dr David A. J. Tyrrell, retired D irector of the British Medical R esearch Council Common Cold U nit, Salisbury, U K; and D r Charles A. Pasternak, Professor of Biochemistry, St George s Hospital Medical School, London, UK, for their unique contributions in support of this research. References 1. Eby, G. A., Davis, D. R. & Halcomb, W. W. (1984). Reduction in duration of common colds by zinc gluconate lozenges in a doubleblind study. 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